The overall goal of this project is to determine whether a rMVA that is being developed for use as an HIV-1 vaccine (MVA/HIV-48) has potential for use as a vaccine for both AIDS and smallpox. MVA was developed for use in immunocompromised individuals toward the end of smallpox eradication. During attenuation in chicken embryo fibroblasts, MVA lost the ability to replicate in primate cells. Despite this, it has shown good promise as a vaccine vector, a promise that likely reflects both its good expression of foreign genes and its loss of many of the immune evasion genes expressed by conventional vaccinia viruses. Because smallpox had been controlled in first world countries by the time that MVA was developed, individuals who were vaccinated with MVA were not exposed to variola, and the efficacy of MVA as a smallpox vaccine was not determined. With the recent bioterrorism events, the development of a vaccine with fewer side effects than the conventional smallpox vaccine has become of high importance. In this application, we seek to compare the anti-vaccinia immune responses raised by the conventional smallpox vaccine Dryvax and MVA/HIV-48 to assess whether MVA/HIV-48 might be able to provide comparable protection to Dryvax. These studies will be done using human samples from employees at the CDC who are receiving the standard human dose of Dryvax and human samples from a phase 1 trial in which a dose escalation will test for the safety of MVA/HIV-48. The dose escalation (1x10'6 to 1x10'8 pfu) will be done with two priming (0 and 4 weeks) and two booster immunizations (12 and 24 weeks), which will allow us to assess if there is a dose/boosting regimen in which MVA/HIV-48 immunizations has comparable immunogenicity to Dryvax. The project will be a collaborative undertaking between researchers at the CDC, the NIAID, and the NIAID HIV Vaccine Trial Network (HVTN ). The focus of this application is the quantification and phenotyping of responding T cells.